1. Field of the Invention
This invention relates generally both to transgenic rats and to animal models of human disease, particularly to transgenic animals which can serve as animal models of human inflammatory diseases. In specific aspects, the invention relates to transgenic rats which have the human HLA-B27 gene inserted into their genome, which develop symptomology not unlike that seen in B27-related disorders in humans.
2. Description of the Related Art
It has become evident that major histocompatibility genes play a role in the development, or propensity for the development, of a number of human inflammatory diseases, including the rheumatic diseases rheumatoid arthritis and the spondyloarthropathies. For example, several human inflammatory diseases of unknown etiopathogenesis are now known to be genetically associated with certain HLA alleles (Tiwari et al., 1985). While in no case has the mechanism underlying such an association yet been elucidated, it is likely that such elucidation will provide new insights into the pathogenesis of a number of hitherto puzzling human diseases.
The disorders commonly classified as spondyloarthropathies share a number of clinical similarities (Calin, 1984; Moll et al., 1974). These disorders can be conveniently separated into two groups, including those with an increased prevalence among B27+individuals and those without an association with B27 (Tiwari et al., 1985). The first group includes ankylosing spondylitis (AS), reactive arthritis, and the spondylitis associated with psoriasis or inflammatory bowel disease; the second group includes the peripheral arthritis associated with psoriasis or inflammatory bowel disease. The inflammatory eye disease, acute anterior uveitis, is strongly associated with HLA-B27, but can occasionally be found with any of these rheumatic disorders in the absence of B27. In none of these disorders is the etiology well understood, although any concept of pathogenesis should include a central role of the HLA-B27 molecule.
Ankylosing spondylitis is an inflammatory disorder of unknown etiology that primarily affects the axial skeleton; peripheral joints and extraarticular structures may also be involved (Taurog and Lipsky 1990). The disease usually begins in the second or third decade; the prevalence in men is approximately three times that in women. It is considered the prototype of the group of disorders collectively referred to as the spondyloarthropathies, which includes ankylosing spondylitis, reactive arthritis, psoriatic arthritis, psoriatic spondylitis, enteropathic arthritis, and enteropathic spondylitis. In Europe, AS is often referred to by the eponyms Marie-Strumpell disease or Bechterew disease.
AS shows a striking association with the presence of the histocompatibility antigen HLA-B27. The disease occurs throughout the human populations of the world in proportion to the prevalence of this antigen. In North American Caucasians, the prevalence of HLA-B27 in the general population is 7%, whereas over 90% of patients with AS have inherited this antigen (Brewerton et al. 1973; Schlosstein et al. 1973). The association with HLA-B27 is independent of disease severity. In large population surveys, 1-2% of adults inheriting HLA-B27 have been found to have AS. In contrast, in families of B27+ patients with AS, 10-20% of adult first degree relatives inheriting HLA-B27 have been found to have the disease. The concordance rate in identical twins is estimated to be 60% or less. These epidemiologic findings indicate that both genetic and environmental factors play a role in the pathogenesis of the disease, and that the genetic factors probably include genes in addition to HLA-B27. Secondary forms of AS occur in individuals suffering from inflammatory bowel disease (enteropathic spondylitis) or psoriasis (psoriatic spondylitis).
Reactive arthritis refers to acute nonpurulent arthritis complicating an infection elsewhere in the body. In recent years, the term has been used primarily to refer to spondyloarthropathies following enteric or ufogenital infections and occurring predominantly in individuals with the histocompatibility antigen HLA-B27. Included in this category is the constellation of clinical findings often referred to as Reiter's syndrome.
Reactive arthritis in human occurs following enteric infection with a variety of gram negative bacteria, including Shigella flexneri, Salmonella species, Yersinia enterocolitica and pseudotuberculosis, and Campylobacter jejuni (Keat, A., 1983; Calin, A., 1984, Toivanen and Toivanen 1988; Taurog and Lipsky 1990). The mechanism by which these infections trigger arthritis is completely unknown. In general, the organisms cannot be cultured from inflamed synovial tissue, although there have been recent observations of bacterial antigens found in synovium following reactive arthritis (Keat et al. 1987; Granfors et al., 1989; Granfors et al., 1990 ).
It is widely assumed that the B27 molecule itself is involved in the pathogenesis of AS and reactive arthritis, although formal proof of this in humans is still lacking. Two types of evidence support this hypothesis. First, genetic and epidemiologic studies provide strong indirect evidence for involvement of B27 in AS (reviewed in Khan, 1988). Second, several studies have suggested antigenic cross-reactivity between the B27 molecule and various enteric organisms, some of which are implicated in triggering reactive arthritis (Raybourne et al., 1988; Chen et al., 1987; Schwimmbeck et al., 1987; Stieglitz, 1989; Yu, 1989).
Elucidation of the molecular basis of the B27-associated diseases is of interest because it might lead to effective prevention and therapy of these relatively common disorders that cause considerable morbidity and disability in otherwise healthy young adults. Such elucidation would likely provide significant insight into other common inflammatory diseases, and it may also contribute to a broader understanding of the function of class I MHC molecules in health and disease. One of the principal reasons behind the inability to provide answers to these important questions has been the unavailability of an animal model for B27 associated disease. Attempts have been made to develop such a model in mice through the transgenic introduction of the B27 gene into the mouse genome (Taurog et al. 1988a; Krimpenfort et al., 1987; Nickerson et al., 1990; Weiss et al., 1990). These efforts were successful in producing transgenic mice which expressed the incorporated HLA-B27 gene. However, despite physiologically normal function of the B27 gene product in mice (Kievits et al., 1987; Taurog et al., 1988a), and despite a reported influence of the B27 transgene on the course of an experimental bacterial infection (Nickerson et al., 1990), no faithful reproduction of any of the features of B27-associated human disease has been reported in B27 transgenic mice (Taurog et al., 1990, Arnold and Hammerling, 1991).
Rheumatoid arthritis is a chronic, immune-mediated inflammatory disease that primarily affects the joints and their supporting structures, although the disease sometimes involves other organs and tissues such as the eyes, lungs, heart, and skin (Harris, 1990). This common disease produces profound morbidity and excess mortality in an estimated 1% of the population (Lawrence et al, 1989).
The alloantigen HLA-DR4 is highly associated with human rheumatoid arthritis (Stastny, 1978). At least eight subtypes of this alloantigen have been identified, including Dw4, Dw10, Dw13.1, Dw13.2, Dw14.1, Dw14.2, Dw15 and Dw "New" (Gao et al., 1990a,1990b). The Dw4 subtype ("DR4,Dw4") is the one most closely associated with rheumatoid arthritis.
Individuals possessing this subtype of DR4 have a risk of developing rheumatoid arthritis about five fold greater than individuals who lack DR4,Dw4 (Zoschke and Segall 1986; Gao et al., 1990b).
As with the B27-related disorders, the availability of an animal model of rheumatoid arthritis would prove instrumental in the development of treatment modalities and protocols, and indeed in the identification of novel agents for the treatment of this important disease. Unfortunately, the development of an animal model closely resembling rheumatoid arthritis has not been previously described.
Various animal models of human diseases have been enabled through the development of transgenic species exhibiting the desired traits. For example, U.S. Pat. No. 4,736,866 to Leder et al. describes technology for the development of transgenic mice having an activated oncogene introduced into the animal's genome. However, while transgene technology has been generally applicable to non-human animals such as mice, cows, pigs, guinea pigs, rabbits, sheep, chickens and even fish, transgenic introduction has proven difficult in some species such as rats, hamsters and guinea pigs.
Due to the widespread use of rats in biomedical research, the development of methods for producing transgenic rats would represent a significant advance. For the study of B27-associated and perhaps even DR4-associated rheumatoid arthritis disease in animals, the advantage of using transgenic rats in addition to mice is at least two-fold; first, arthritis is more readily inducible in rats than in mice; and, second, there are more conventional experimental models of arthritis in rats than in mice in which to study the influence of the B27 gene.
The development of an animal model of B27-associated or DR4associated disease in which all of these difficulties are circumvented would represent a significant advance in the understanding of this group of human disorders. For example, such models could provide a direct means for screening new anti-arthritic compounds or compounds which have the ability to treat symptoms of the various diseases, such as in the identification of new anti-inflammatory, anti-arthritic agents, anti-psoriatic agents, agents for the treatment of spondylitis, and the like. Moreover, on a scientific level, such a model could answer many of the questions which have arisen from our lack of information regarding these diseases.